T. douronensis

POPULATION GENETIC STRUCTURE OF TWO
MAHSEER (Tor tambroides BLEEKER AND Tor
douronensis VALENCIENNES: CYPRINIDAE) IN
MALAYSIA INFERRED FROM ANALYSIS OF
MICROSATELLITE LOCI
Yuzine bin Esa, Siti Shapor Siraj, Siti Khalijah
Daud, Khairul Adha A. Rahim, Tan Soon Guan
Universiti Malaysia Sarawak
Universiti Putra Malaysia
INTRODUCTION
•Mahseer –locally known as ikan Kelah, Semah, Empurau, Pelian
•Belong to family Cyprinidae
•One of the most important freshwater fish – food, aquaculture
potential, aquarium, game fish
•Habitat – upper stream with clean, fast flowing water, rocky, high
oxygen
Tor douronensis
Tor tambroides
Median lobe
short
long
PROBLEM STATEMENTS
Population structure of mahseer
•Population size – low and highly fragmented due to
anthropogenic/enviromental disturbances, uncontrolled fishing
activities
•May affect their genetic variabilities; low genetic diversity, high genetic
structuring
OBJECTIVES
To examine the genetic diversity and population structure of T.
douronensis and T. tambroides natural populations using
microsatellite loci.
METHODOLOGIES
Samples of mahseer:
•152 T. tambroides samples: Negeri Sembilan (N=20), Pahang (N=17), Perak (N=19),
Kelantan (N=20), Endau-Rompin (N=61)), Batang Ai (N=5), Batang Baleh (N=5) and Ulu
Limbang (N=5).
•77 T. douronensis samples: Batang Ai (N=33),Layar/Spak (N=5), Bario (N=9), Ba Kelalan
(N=8), Ulu Limbang (N=13) and Sabah (N=9).
•Morphologically identified following Mohsin & Ambak (1983), Kottelat et al. (1993)
Locations of sample collections
Microsatellites:
i) 14 microsatellites loci developed by Nguyen et al. (2007).
ii) 5 polymorphic loci in T. tambroides (MFW7, Barb37, Barb59, Barb62, Bgon13)-cross species
iii) 4 polymorphic loci in T. douronensis (MFW7, Barb37, Barb62, Bgon13)-cross species
Samples
DNA extraction
Polymerase Chain Reaction (PCR)
Gel electrophoresis
Staining (Ethidium bromide)
Visualization and photograph
Band/allele scoring
Statistical analyses
Statistical analyses:
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Genotyping errors (i.e. null allele) –MICRO-CHECKER
Allelic richness
Observed heterozygosity (Ho) & Expected heterozygosity (He)
Hardy-Weinberg Equilibrium (HWE) test
F-statistics
Population subdivision (FST)
Analysis of Molecular Variance (AMOVA)
Genetic distances between samples
Assignment test (Bayesian method),
Number of clusters K (Bayesian method)
Results Highlights
Microsatellite polymorphism:
•MICRO-CHECKER detected presence of null alleles at three loci (Tt1.B01, Barb 37 and
Barb62) in T. tambroides and at one locus (Barb37) in T. douronensis, as suggested by
the general excess of homozygotes for most allele size classes.
•Allelic richness per locus ranging between 1.2110 (Bgon13) and 4.6670 (Tt1.B02) in T.
tambroides and between 1.2820 (MFW7) and 4.0000 (Barb62) in T. douronensis.
•Expected heterozygosity within each populations ranged between 0.0163 (MFW7 of
Endau-Rompin) and 0.7900 (Tt1.B02 of Negeri Sembilan) in T. tambroides and between
0.0588 (MFW7 of Negeri Sembilan) and 0.6600 (Barb62 of Pahang) in T. douronensis.
Results Highlights
Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium
• HWE tests showed that 6 (40.0%) of the 15 locus –deviated in T. tambroides while
only 2 (15.3%) out of the 13 locus were deviated in T. douronensis.
•In all populations, FIS values were significantly different from zero (p< 0.05) in both
species (indicating heterozygote deficiencies) except in T. tambroides population of
Endau-Rompin.
•For T. tambroides, 26 (20.5%) pairwise comparisons were in linkage disequilibrium.
After pooling all populations, 12 comparisons were highly significant.
•For T. douronensis, 10 (10.3%) pairwise combinations showed significant linkage
disequilibrium. Pooling of all populations produced only five significant linkage
disequilibrium.
Results Highlights
Genetic differentiation among populations and species
•AMOVA results revealed that majority of the variance in T. tambroides (83.94%) was
from intra-population variations, and only 13.13% of total variance resulted from interpopulation differentiation.
•In T. douronensis, 82.71% of the total variance was also from intra-population
variations, and only 13.79% of the total variance resulted from inter-population
differentiation.
•Within T. tambroides, 22 (78.5%) out of 28 pairwise estimates of FST showed
significant genetic differentiation with the Kelantan population showed the highest
degree of differentiation from all other populations (FST = 0.1811-0.6494, p< 0.05).
•Within T. douronensis, only 4 (20.6%) out of 15 pairwise estimates of FST showed
significant differentiation with the highest value was between Batang Ai population
and Ulu Limbang population (FST = 0.3533, p< 0.05).
Results Highlights
Genetic differentiation among populations and species
•Pairwise estimates of genetic distances computed by Nei (1978) were lower among T.
douronensis populations compared with among T. tambroides populations.
•The highest genetic distances within T. tambroides was between Kelantan population
and Perak population (0.1709) while the highest genetic distances within T.
douronensis was between Batang Ai and Layar (0.0484).
•Results of the assignment tests (Piry et al., 2004) using GENECLASS2 showed that on
average 42.8% (66 out of 152) and 52.6% (40 out of 76) of the individuals were
correctly assigned to their original sampling sites in T. tambroides and T. douronensis
respectively.
•The Perak population presented the highest percentage of correctly assigned
individuals (72.6%) in T. tambroides while the Batang Ai population produced the
highest number of correctly assigned individuals (97.3%) in T. douronensis.
T. tambroides
Pairwise genetic distances between populations (below diagonal)
Pairwise FST values between populations (above diagonal)
T. douronensis
Pairwise genetic distances between populations (below diagonal)
Pairwise FST values between populations (above diagonal)
Results Highlights
Genetic differentiation among populations and species
•Bayesian cluster analysis performed with STRUCTURE (Pritchard et al., 2000) showed
that the most likely K value identified was K = 3 for T. tambroides and K = 2 for T.
douronensis.
•For T. tambroides, the three clusters are (i) Cluster I: Negeri Sembilan, Pahang and
Perak, (ii) Cluster II: Kelantan and (iii) Cluster III: Batang Ai, Baleh and Ulu Limbang of
Sarawak.
•For T. douronensis, the two clusters are (i) Cluster I: Batang Ai and (ii) Cluster II: other
T. douronensis populations consists of Layar, Bario, Ba Kelalan, Ulu Limbang and Sabah.
•The UPGMA dendrogram generated three clusterings within the T. tambroides
populations, similar to the clusters identified by STRUCTURE.
•However, three clusters were constructed within T. douronensis populations, as
opposed to the two clusters identified by STRUCTURE.
•The three UPGMA clusters were: (i) Batang Ai population, (ii) Layar, Bario and Ba
Kelalan populations, and (iii) Ulu Limbang and Sabah populations.
T. tambroides
The 3 clusters identified using STRUCTURE
T. douronensis
The 2 clusters identified using STRUCTURE
UPGMA dendrogram of both mahseer
DISCUSSION
1) Genetic diversity and population differentiation
•Microsatellite analyses reveal a higher degree of population differentiation among
extant populations of T. tambroides (FST: 0.0011 to 0.6494, genetic distances: 0.2% to
17.1%) compared with the T. douronensis populations (FST: 0.0057 to 0.3533, genetic
distances: 0.0% to 4.8%).
•The low population differentiation of T. douronensis found in this study is not
consistent with Nguyen (2007) who found high population subdivision among 13 T.
douronensis populations in Sarawak.
•Nevertheless, our current results generally supported the indication of two welldefined clusters (the northeastern and the southwestern clusters).
DISCUSSION
2) Comparisons of microsatellite data with previous mitochondrial
sequences data
•The patterns of genetic variations differed between results generated using
microsatellites and mitochondrial DNA in both mahseer.
• Microsatellites found high levels of within (intra) population variations but
mitochondrial results found high levels of among (inter) populations differentiation.
•Two suggested hypotheses:
(i) may reflect the varying influence of genetic drift on mitochondrial and nuclear
genomes.
(ii) small sample sizes in most of the populations, where some of the alleles could not
be detected, thus reducing their overall level of genetic variations.
DISCUSSION
3) Conservation and management implications
•Conservation unit concept of Moritz (Moritz 1994a, b; Moritz et al., 1995) which
defines an Evolutionary Significant Unit (ESU) as requiring reciprocally monophyly of
mitochondrial DNA genes and significant differentiation at nuclear alleles.
•T. tambroides and T. douronensis be recognized as different ESUs due to their
reciprocally monophyletic status identified by mitochondrial data and significant
differentiation at microsatellites loci.
•Management by river systems (Management Units[MU]).
•Populations/rivers with unique genetic (mitochondrial haplotype or nuclear allele)
markers (i.e. Layar river) or shows very low level of genetic variability should be given
priority
•Habitat protection – crucial for continuous survival of mahseer population
CONCLUSION
The informations on the levels and distribution of genetic variability
produced by microsatellite (and previous studies using mitochondrial
DNA) markers should comprise a key component of the mahseer
conservation and management plan, and is also important for mahseer
evolutionary persistence.
Thank You
Terima
kasih